Patent Application
20230151386
The present invention relates to apparatus for achieving improved transfection efficiency and/or protein expression and a method of use thereof. The apparatus can also be used to improve protein expression in cells and a method of use thereof.
Although the following description makes reference to how apparatus for allowing improved transfection of cells can be used for the purposes of gene therapy, it will be appreciated by persons skilled in the art that the present invention can be used for any purpose or application where the transfection of cells is required, such as in the production of viral vectors, gene therapy or modification, protein expression, autologous cell therapy and/or the like. It will also be appreciated that the apparatus and methods of the present invention can be undertaken in respect of in vitro cells, ex vivo cells and/or in vivo cells.
Transfection is a process by which nucleic acid is introduced into eukaryotic cells. Transfection can be stable, in that the transfected nucleic acid may be continuously expressed and is passed on to daughter cells. Alternatively, transfection can be transient, in that the transfected nucleic acid is only expressed for a short period of time following the transfection and is not passed on to daughter cells. The use of either type of transfection in the field of gene therapy is well known [1] and focuses on the utilization of the therapeutic delivery of nucleic acid into a patients cells to act as a drug for the treatment of a disease. For example, the purpose can be to replace faulty genes in a patient that, if not treated, could lead to the patient suffering from gene related and inherited conditions. In the laboratory setting, immortal cell lines are often transfected with an exogenous gene, typically in the form of a plasmid. Following transfection, the successfully transfected cells will express the exogenous gene.
In a transient transfection, an exogenous gene (typically encapsulated in a carrier such as polyethylenimine (PEI)) will be introduced to a population of cells. A portion of these cells will be successfully transfected, and will begin to express the exogenous gene. After a short period of time, the level of expression will fall, and the cells are typically processed or otherwise discarded at this point.
In a stable transfection, the cells are transfected as above. A portion of the cells will have integrated the exogenous gene in a stable manner. The stably transfected cells can be isolated and selected from the population of cells based upon expression of the exogenous gene, and these cells propagated to produce an immortal cell line expressing the exogenous gene over a longer period of time.
Transfection efficiency (i.e. the rate at which cells are successfully transfected with an exogenous gene) is typically low in the prior art methods. Multiple strategies have been adopted to try to increase the transfection efficiency of cell lines (e.g. electroporation, specialised reagents for transfection, and others). What is needed is apparatus and a method for further improving transfection protocols in order to improve the transfection efficiency of any given transfection protocol.
A recent development has been to manipulate the genetic sequence of a patients own immune cells to transform them into cells that will recognise and attack specific cancerous cells within the patients body [2]. Approaches where a patients cells are genetically manipulated is known as ‘gene therapy’. Once promising avenue of gene therapy for treatment of cancer is the genetic manipulation of T-cells such that they express chimeric antigen receptors which allow the T-cells to target cancerous tissue growth more effectively. Such cells are known as chimeric antigen receptor T-cells (“CAR-T” cells) and the therapy is known as “CAR-T cell therapy”. The immune cells are first removed from the patients body and then undergo a transfection process ex vivo that converts the cells to cancer-seeking killer cells. The transfected cells are then re-administered to the patient to treat their cancer. The transfection of these cells is typically achieved using an approach involving associating the exogenous genetic material with a carrier molecule, such as a nanoparticle or a liposomal carrier.
An example of a conventional transfection process includes the step of encapsulating target DNA in a phospholipid, bilayer vesicle or liposome that is then administered into a eukaryotic cell [3]. As the liposome is formed of phospholipid, the liposome has an affinity for eukaryotic cell membranes that, likewise, have a phospholipid bilayer, and so there is fusion of these systems. External DNA can therefore be transferred via this fusogenic mechanism into the eukaryotic cell and become extrachromosomal genetic information for the cell. A simple conventional transfection process involves encapsulating the exogenous nucleic acid (e.g. DNA plasmid containing the gene of interest) in a cationic polymer (PEI) [6]. While there are potentially significant advantages of such processes, these conventional processes are slow and have a poor transfection efficiency. The low transfection efficiency of these methods makes then wasteful and time consuming, thus expensive.
It is therefore an aim of the present invention to provide apparatus that improves transfection efficiency and/or protein expression in eukaryotic cells that overcomes the abovementioned problems.
It is a further aim of the present invention to provide a method of improving transfection efficiency and/or protein expression in eukaryotic cells.
It is a further aim of the present invention to provide transfection enhancing apparatus and/or to a method of use thereof.
It is a yet further aim of the present invention to provide apparatus that improves the effectiveness of gene therapy and/or therapeutic treatment in animals or humans.
It is a yet further aim of the present invention to provide a method of improving the effectiveness of gene therapy and/or therapeutic treatment in animals or humans.
It is a yet further aim of the present invention to provide apparatus that improves the production of viral vectors and/or a method of use thereof.
It is a yet further aim of the present invention to provide apparatus that improves protein expression in human and/or animals cells and/or a method of use thereof.
A further aim of the present invention is to allow the speed of preparation and/or application of transfection material to be improved and a yet further aim is to allow an increased yield of transfected cells.
It is a yet further aim of the present invention to provide apparatus and/or method of use which allows for the delivery of an agent, drug and/or therapeutic treatment through the skin of a patient in a more targeted and efficient manner at lower cost.
It is a further aim of the present invention to provide apparatus and/or method of use which can be used to provide the delivery of an agent, drug and/or a therapeutic treatment to a patient which allows the apparatus to be easily portable and/or used in a patients home.
According to a first aspect of the present invention there is provided a method of improving transfection efficiency in eukaryotic cells, said method including the steps of:
The Applicants have surprisingly found that the administration of pulsed electromagnetic (PEM) signals before, during and/or after transfection significantly increases the transfection efficiency and/or protein expression yield created by the transfection process. The transfection rate is significantly improved and allows for the enhanced frequency of transfected cells containing the agent and/or exogenous nucleic acid. Thus, the present invention provides a non-invasive, non-chemical approach to improving cell viability, gene transfer, transfection rate and/or protein production. The present invention enhances the transportation of extra-cellular material or agent from an environment external to a cell to an internal environment in the interior of the cell.
The term “pulsed electromagnetic signals” used herein is preferably defined as a sequence or pattern of signals in the electromagnetic spectrum range that change in amplitude from a base line to a higher or lower value, followed by a return to the base line or a return substantially to the base line. Further preferably the change in signal amplitude is rapid and transient and occurs in a repeating sequence. In one example, the base line represents an absence of electromagnetic signals being emitted from an electromagnetic signal source or transmission means. Preferably the base line is considered to be a rest or relaxation period for the cells and/or pulsed electromagnetic signals.
Preferably the method can take place entirely in-vitro, entirely in-vivo, or partially in-vitro and partially in-vivo. For example, the eukaryotic cells could be transfected in vitro and used for one or more purposes or applications in vitro. In a further example, the eukaryotic cells could be extracted from a patient, transfected in-vitro and then re-introduced back into the patient (this is interchangeably referred to as an “ex vivo” method. Alternatively, the transfection complex could be injected or otherwise transported into a patient and the patients cells could be transfected in-vivo.
Preferably the agent in the transfection mixture is any agent suitable for transfection and/or any or any combination of nucleic acid, a pharmaceutical and/or therapeutic agent or compound, an agent of therapeutic and/or pharmaceutical interest, a small molecule or small molecular material of less than 5 Kilodaltons, a large molecule or large molecular material greater than or equal to approximately 5 Kilodaltons, one or more proteins, vaccine, one or more antibodies, an organic agent and/or the like.
The term ‘pharmaceutical and/or therapeutic agent or compound’ preferably refers to compounds which are deployed or being developed for deployment into the clinic, which have a defined medicinal effect.
The term ‘agent of therapeutic and/or pharmaceutical interest’ preferably refers to compounds that have been developed for use and/or are being investigated for use in research and/or in the clinic. These agents or compounds may have a known mechanism of action, but the clinical suitability and relevance may not have been demonstrated or investigated. In some embodiments, the mechanism of action of these agents or compounds may not yet have been uncovered. Regardless, the underlying mechanism of the present invention allows superior intracellular delivery of these agents or compounds.
In one aspect, there is provided a method of intracellular delivery, said method comprising:
In some embodiments, the agent is or comprises a nucleic acid, and transfection refers to the process by which the nucleic acid is introduced into the one or more eukaryotic cells, thus causing the cell(s) to express an exogenous nucleic acid (for example an exogenous RNA, DNA, RNA/DNA hybrid or a gene encoded by, or a protein expressed from, said nucleic acid). In other embodiments, where the agent does not comprise a nucleic acid, the word transfection is used interchangeably with the term ‘intracellular delivery’, and in this context means that the agent is delivered across the cell membrane into the cytoplasm of the one or more eukaryotic cells. In some embodiments, the method, particularly when the agent does not comprise a nucleic acid, can thus be considered to be a method of trans-membrane delivery.
In any aspect or embodiment described herein, unless otherwise apparent, the method may be considered to be a method of transfection, intracellular delivery or trans-membrane delivery.
In any aspect or embodiment described herein, unless otherwise apparent, the transfection mixture can be described as a first mixture, and/or the transfection complex can be described as a second mixture. The delivery to the cells may be considered to be trans-membrane, intracellular or intra-cytoplasmic delivery.
Preferably the agent is associated with the at least on amphiphilic construct in that it is contained within the amphiphilic construct, it forms a complex with the amphiphilic construct, it is contained on the amphiphilic construct, it is bonded to the amphiphilic construct and/or the like.
In one embodiment the eukaryotic cells could include any or any combination of adherence cells, suspension cells, blood cells, lymphocytes, granulocytes, T-cells and/or the like.
In some embodiments, the eukaryotic cells are suspended in solution, adhered to a substrate, or a mixture of both suspended and adhered cells.
In some embodiments, the eukaryotic cells are immortal cells or cells derived from an immortal cell line. For example, Chinese Hamster Ovary (CHO) cells, Human Embryonic Kidney (HEK) cells, Human Colon Tumour (HCT) 116 cells, or Jurkat E6 cells.
In some embodiments, the eukaryotic cells are the cells in or derived from the tissue of a human or animal subject. For example, cells may have been extracted from a subject to be transfected and then reintroduced to the subject. In some embodiments, the eukaryotic cells are derived from the blood of a subject. In some embodiments, the eukaryotic cells are T-cells, lymphocytes, granulocytes, macrophages and/or other white blood cells. In some embodiments, the T-cells are any or any combination of helper T-cells or cytotoxic T-cells. In some embodiments, the T-cells comprise CD4+ cytotoxic T lymphocytes and/or CD8+ cytotoxic T lymphocytes.
One exemplary use of the apparatus and method of the invention is adoptive T-cell therapy (ACT), involving the generation of so called ‘CAR-T’ cells. In such a technique, the apparatus and/or method are used on T-cells derived from a subject. The cells are cultured and transfected in vitro to express the chimeric antigen receptor, and then expanded in vitro prior to being reintroduced into the patient.
The present apparatus and/or method improves the transfection efficiency and thus provides a higher yield of CAR-T cells.
In some embodiments, the method may not be a method of treatment or surgery carried out on the human or animal body. In some embodiments, the method may not be a method for modifying the germ line genetic identity of human beings.
Preferably the method includes the step of mixing the nucleic acid or agent with the at least one amphiphilic construct to form the transfection mixture. Once the nucleic acid or agent is associated with the amphiphilic construct it forms the transfection mixture.
In one embodiment the at least one amphiphilic construct can include or consist of any or any combination of at least one liposomal material or vehicle, at least one pegylated liposomal material or vehicle, a micelle, a construct having a phospholipid bilayer, a cationic polymer, polyethylenimine (PEI) and/or the like.
For example, the cationic polymer can be Turbofect™.
Preferably the nucleic acid is deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or comprises a combination of DNA and RNA (for example, DNA/RNA hybrid oligonucleotides). When the nucleic acid is RNA, it can be mRNA, tRNA, siRNA, miRNA and/or the like.
In one embodiment, the nucleic acid is or includes one or more expression vectors. For example, the one or more expression vectors could be one or more DNA plasmids comprising one or more exogenous genes intended for expression in one or more eukaryotic cells.
In one embodiment, when the agent is nucleic acid, the transfection process results in stable expression, in that the transfected nucleic acid in the transfected cells is continuously expressed and is passed on to daughter cells.
In one embodiment, when the agent is nucleic acid, the transfection process results in transient expression, in that the transfected nucleic acid is only expressed for a relatively short period of time and is not passed on to daughter cells.
Preferably the step of directing pulsed electromagnetic signals takes place at room temperature (such as for example 20° C.) or takes place in an incubator that can be set at temperatures above room temperature or in a patients body (such as for example at 37° C.).
In one embodiment the step of directing pulsed electromagnetic signals takes place for a pre-determined time period. In one example, the time for which the cells receive the pulsed electromagnetic signals is approximately 15 minutes or up to 15 minutes when directed at the transfection mixture in step a) prior to creating the transfection complex. However, it will be appreciated that longer or shorter time periods could be used if required.
In one embodiment the pre-determined time period for which the cells receive the pulsed electromagnetic signals is approximately at or between approximately 1-4 hours when directed at the transfection complex in step c) to form the transfected cells and/or after the transfection step, and further preferably approximately 3-4 hours. However, it will be appreciated that longer or shorter time periods could be used if required. For example, in one embodiment the pre-determined time period can be up to 16 hours, or up to 24 hours.
In one embodiment the transfection is reverse transfection (i.e. the eukaryotic cells are introduced into the transfection mixture).
In one embodiment the transfection is forwards transfection (i.e. the transfection mixture is introduced into the eukaryotic cells).
Preferably the pulsed electromagnetic signals are generated by one or more electronic devices.
Preferably the one or more electronic devices include transmission means for generating and/or transmitting the pulsed electromagnetic signals therefrom in use.
Preferably the transmission means includes one or more electronic transmission chips, the one or more electronic transmission chips arranged to generate, emit and/or transmit one or more pulsed electromagnetic signals in use.
In one embodiment reference to the transmission means or one or more electronic transmission chips could include one or more transmitters, at least one transmitter and at least one receiver, or one or more transceivers. Thus, in one example, the pulsed electromagnetic signals could be transmitted from a central location or a master transmitter and could be received by one or more remote and/or slave receivers and/or transceivers for subsequent re-transmission or emission therefrom.
In one embodiment the electronic device has a single transmission means or electronic transmission chip. Such a single transmission means or electronic transmission chip is sufficient to provide a pulsed electromagnetic signal to a tissue culture plate in one example. In one exemplary embodiment, a single transmission means or electronic transmission chip is provided attached or integrated into a bioreactor containing one or more suspended cells. Such a bioreactor operates by stirring the suspension with a stirrer, and as such the cells suspended, typically in media, will pass by the transmission means or electronic transmission chip and thus be exposed to the pulsed electromagnetic signal of the present invention.
In one embodiment the electronic device has two or more transmission means or electronic transmission chips. Preferably the two or more transmission means or electronic transmission chips are arranged a pre-determined spaced distance apart from each other in the electronic device.
Preferably the pre-determined spaced distance apart is such so as to provide one or more items or material being pulsed with the electromagnetic pulsed signals sufficient signal strength to achieve a desired effect (i.e. of increasing transfection efficiency) and/or to provide an even or substantially even distribution of electromagnetic radiation/signals in use.
Preferably the electronic device has a plurality of transmission means or electronic transmission chips arranged in a pre-determined pattern and/or array.
Whilst a single transmission means or electronic transmission chip is sufficient to provide the advantageous properties of the invention, it has been found that having a plurality of transmission means or electronic transmission chips allows the pulsed electromagnetic signal to be delivered across a broader range of surface areas whilst still maintaining a maximal effect. Applicants have found that having a transmission means or electronic transmission chip evenly distributed such that there is at least one chip per 18.5 cm2 provides sufficient coverage for the optimal effect.
In some embodiments, the apparatus comprises one or more transmission means or electronic transmission chips. In some embodiments, the apparatus comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more transmission means or electronic transmission chips.
In some embodiments, there is one transmission means or electronic transmission chip per approximately 105 to 115 cm2 of a surface of the housing of the apparatus or a surface of an item as defined herein, and preferably approximately 110 cm2 of a surface of the housing of the apparatus or a surface of an item as defined herein.
In some embodiments, there is one transmission means or electronic transmission chip per approximately 50 to 60 cm2 of a surface of the housing of the apparatus or a surface of an item as defined herein, and preferably approximately 55 cm2 of a surface of the housing of the apparatus or a surface of an item as defined herein.
In some embodiments there is one transmission means or electronic transmission chip per approximately 25 to 30 cm2 of a surface of the housing of the apparatus or a surface of an item as defined herein, and preferably approximately 27.5 cm2 of a surface of the housing of the apparatus or a surface of an item as defined herein.
In some embodiments there is one transmission means or electronic transmission chip per approximately 15 to 20 cm2 of a surface of the housing of the apparatus or a surface of an item as defined herein, and preferably approximately 18.5 cm2 of a surface of the housing of the apparatus or a surface of an item as defined herein.
In some embodiments, there is one transmission means or electronic transmission chip per approximately 10 to 15 cm2 of a surface of the housing of the apparatus or a surface of an item as defined herein, and preferably approximately 12.2 cm2 of a surface of the housing of the apparatus or a surface of an item as defined herein.
The items as defined herein preferably comprise cell culture plates, flasks, roller bottles, and other vessels known to the skilled person. For example, standard laboratory microplates as defined below, T25, T75, T125, T175, T225, and larger cell culture plates. The one or more transmission means or electronic transmission chips are set a pre-determined space apart according to the surface area of such vessels placed on the device in use, and/or based upon a surface of the housing of the apparatus.
In an exemplary embodiment, six transmission means or electronic transmission chips are provided in the apparatus upon which a standard laboratory microplate is positioned. These standard laboratory microplates are provided as 6-well, 12-well, 24-well, 48-well, 96-well, 384-well, and 1536 well plates (and above). These microplates are generally of a standardized size, with dimensions of approximately 128 mm in length by 85 mm in width, thus giving the plate a surface area of approximately 110 cm2. Thus, in the exemplary embodiment, the 6 transmission means or electronic transmission chips can be evenly spaced to provide an optimal pre-determined space for providing any of these plate types with a pulsed electromagnetic signal according to the present invention. In one example, the electronic device includes six transmission means or electronic transmission chips. Preferably the six transmission means or electronic transmission chips are arranged a pre-determined distance apart from each other such that when a 24 well plate is located in, on or relative to the electronic device in use, each transmission means or chip is able to emit sufficient strength electromagnetic signals and/or is directed to 4 wells of the plate.
Further preferably the transmission means or transmission chip is located adjacent to the 4 wells of the 24 well plate in a central or substantially central position.
In one embodiment, where more than one transmission means or electronic transmission chip is required, the spacing of the plurality of transmission means or electronic transmission chips must be optimised. In order to achieve an optimal pre-determined space between each transmission means or electronic transmission chips, the transmission means or electronic transmission chips should be positioned at a distance equal or substantially equal to half the wavelength of the electromagnetic radiation frequency being used. Preferably this distance should be considered to be relevant in any plane of orientation or two or more transmission means or electronic transmission chips being used together as part of the apparatus. For example, if the wavelength is 12.4 cm, the transmission chips should be placed approximately 6.2 cm apart to produce an optimal electromagnetic field when in use.
In one example, the pre-determined spaced distance=wavelength/2.
In one example, the pre-determined spaced distance in the X-axis and/or Y-axis is half the wavelength between each transmission means or electronic transmission chip in an evenly spaced grid. Such an arrangement minimises the risk of destructive interference.
In one embodiment the electronic device includes a housing and the one or more transmission means or transmission chips are located in said housing.
Preferably the housing includes at least one flat or planar surfaces to allow the housing to be located in a stable manner with respect to the one more items receiving the pulsed electromagnetic signals in use. Alternatively, the housing can include one or more curved or non-planar surfaces to allow the housing to be located in a stable manner with respect to one or more items receiving the pulsed electromagnetic signals in use.
In one example, at least one surface of the housing includes one or more recesses for the location of the one or more items receiving the pulsed electromagnetic signals in use.
In one example, the electronic device is referred to as a transfection plate for use in a laboratory.
In one embodiment the housing includes a base surface for allowing the housing to be supported directly or indirectly on a surface in use. Further preferably the housing includes an upper surface opposite to the base surface. Preferably the upper surface is the surface on which the one or more items receiving the pulsed electromagnetic signals can be positioned in use.
In one example, the one or more items can be cell culture plates or flasks known to the person skilled in the art in which eukaryotic cells may be cultured.
In one embodiment the electronic device and/or housing is attachable to an external surface of a container, reactor vessel and/or the like. For example, the electronic device and/or housing can be attachable via one or more attachment means or device including any or any combination of one or more screws, nuts and bolts, magnets, ties, clips, straps, inter-engaging members, adhesive, welding and/or the like.
Preferably the upper surface of the housing and/or the distance between the transmission means and the one or more items receiving the pulsed electromagnetic signals when located on, in or relative to the housing or electronic device in use is approximately 25 cm or less, 20 cm or less, 15 cm or less, 10 cm or less or 5 cm or less. Further preferably the distance is approximately 1 cm.
Preferably the pulsed electromagnetic signals are provided in a pre-determined sequence of pulses.
In one embodiment the electronic device is arranged to transmit the pulsed electromagnetic signals at a frequency in the range of approximately 2.2-2.6 GHz and, further preferably the pulsed electromagnetic signals are transmitted at a frequency of approximately 2.4 GHz+/−50 MHz or more preferably 2.45 GHz+/−50 MHz.
In one embodiment the electronic device is arranged to transmit the pulsed electromagnetic signals at a frequency within the range of the industrial, scientific and medical radio frequency band (ISM band) of 2.4 to 2.4835 GHz, preferably 2.45 GHz+/−50 MHz.
Preferably the pulsed electromagnetic signals are pulsed at a frequency of approximately 50 Hz or less, further preferably approximately 25 Hz or less, and yet further preferably approximately 15 Hz or less.
Preferably each pulse of the pulsed electromagnetic signals lasts for between approximately 1 ms-20 ms. Further preferably each pulse lasts for approximately 1 ms.
Preferably the time period between pulses (also referred to as the “rest period” or “relaxation period”) is approximately 66 ms or less.
Preferably the duty cycle of the pulsed electromagnetic signals is less than 2%.
In one embodiment the transmission power provided by each transmission means or chip in the electronic device is 2 dBm-+4 dBm, approximately 1 mW, approximately 2 mW or approximately 2.5119 mW.
In one embodiment the pre-determined frequency of the pulsed electromagnetic signals is approximately 2.2-2.6 GHz, 2.4 GHz+/−50 MHz or 2.45 GHz+/−50 MHz, the pre-determined pulse rate is approximately 15 Hz or has a duty cycle of less than 2%, and the pre-determined power is +2 dBm-+4 dBm, 1 mW, 2 mW or 2.5119 mW.
Without wishing to be bound by theory, use of electromagnetic waves or signals used in the apparatus or methods of the present invention are thought to be sufficient to rotate H2O periodically around its dipole with relatively long rest or relaxation periods. The periodic rotation of H2O is thought to interrupt hydrogen bonding in the phospholipid bilayer and/or amphiphilic constructs. This periodic or intermittent low energy perturbation of the cell membranes is thus thought to stimulate increased interaction with some molecules, cell membranes and/or amphiphilic constructs and their environment, such as for example, the nucleic acid or agent associated with the amphiphilic construct. This is thought to enhance the transport of agents across the cell membrane, leading to an increased uptake of the one or more agents such as nucleic acids, peptides, small molecules and other agents by the one or more eukaryotic cells. Thus, it can be seen that the transfection and/or intra-cellular delivery process according to the present invention can be significantly improved using very low energy electromagnetic waves or signals. The relatively long rest or relaxation period between the pulses of the pulsed electromagnetic signals is thought to be sufficient to maintain cellular integrity. Thus, in the context of the present invention, the use of pulsed electromagnetic signals, waves or fields, is thought to provide an improved transport of molecules across the cell membrane, leading to a more efficient transfection and/or intracellular delivery of agents as defined earlier.
Preferably the pulsed electromagnetic signals are transmitted using Gaussian Frequency Shift Keying (GFSK) between 0.45 and 0.55.
Preferably the pulsed electromagnetic signals are radio frequency (RF) data signals.
Preferably the pulsed electromagnetic signals is a digital sequence of pulsed electromagnetic signals.
Preferably the radio frequency signals utilize the Bluetooth LE (BLE) protocols advertising feature. Preferably the advertising RF signals are on channels 37, 38 and 39 corresponding to frequencies 2402 MHz, 2426 MHz, 2480 MHz respectively.
Preferably the pulsed electromagnetic signals are directed towards aqueous media consisting of or including the transfection mixture, transfection complex and/or a post transfection complex.
In one embodiment the electronic device includes power supply means for supplying electrical power to the device in use. Preferably the power supply means includes a mains electrical power supply, one or more batteries, power cells, one or more rechargeable batteries, electrical generator means and/or the like.
In one embodiment the electronic device includes control means for controlling operation of the electronic device and/or transmission means in use.
In one embodiment the electronic device includes one or more circuit boards. Preferably the transmission means can be provided on the one or more circuit boards, typically in the form of an integrated circuit, and/or other components, such as for example memory means, are located.
In one embodiment the electronic device includes memory means, such as a memory device, data storage device and/or the like.
Preferably the other components of the electronic device includes one or more components required for the selective operation of the apparatus and, when active, the controlled operation of the same to generate the pulsed electromagnetic signals. For example, user selection means can be provided on the device to allow user selection of one or more conditions, operation and/or one or more parameters of the device in use; display means to display one or more settings, options for selection and/or the like.
In one embodiment the said further components or power supply means include one or more power cells and the same may all be contained within the housing.
In one embodiment the housing of the electronic device is provided in a form which allows the same to be engaged with and/or located with respect to a container in which the material and/or one or more items which is to be exposed to the electromagnetic signals is located in use.
In one embodiment the control means includes an option to allow the user to select any or any combination of the signal frequency, signal strength, signal power, signal pulse rate, time period of signal pulsing, and/or the like of the said pulsed electromagnetic signals. In one embodiment the selection of the frequency, strength, power, pulse rate, time period of pulsing, other parameters and/or the like may be made with respect to the particular form of the material and/or one or more items which is to be exposed to the pulsed electromagnetic signals in use, the quantity of said material, the dimensions of the container with respect to which the apparatus is located for use and/or other parameters.
It has been found the cells exposed to pulsed signals like those of the present invention provide a uniform or substantially uniform distribution or dispersion of cells during transfection in vitro, in contrast to transfection where no pulsed technology is used and clumping of cells has been observed.
According to an aspect of the present invention there is provided apparatus for providing improving transfection efficiency in eukaryotic cells, said apparatus including a housing, transmission means located in said housing and arranged to transmit pulsed electromagnetic signals provided at any or any combination of a pre-determined frequency, at a pre-determined pulse rate, or a pre-determined power in use, control means for controlling operation of at least the transmission means in use, and power supply means for providing electrical power to the transmission means and/or control means in use.
Preferably the one or more pre-determined parameters of the apparatus can be pre-set by the manufacturer of the apparatus and/or can be user selectable depending on the users requirements.
Preferably the control means are used to allow user selection of one or more of the user selectable pre-determined parameters.
In one embodiment the apparatus is arranged to be directly or indirectly worn on or adjacent the skin of a person in order to allow the pulsed electromagnetic signals to be directed towards an area of the persons body in use for improving a transfection process in the persons body. In this embodiment the apparatus is preferably a wearable device.
In one embodiment, attachment means can be provided on and/or associated with the apparatus to allow detachable attachment to, or relative to, the exterior of a users skin or body, the interior and/or exterior of a garment or item worn by the user in use and/or the like for improving a transfection process taking place in the persons body.
In an exemplary embodiment, the apparatus is a wearable device, for example an armband, and the armband is placed directly on the site of injection of, for example, a DNA or RNA vaccine administered to a patient.
In an exemplary embodiment, there is a method for administration of a vaccine comprising injecting the vaccine into a subject, and then placing the apparatus of the invention on the site of injection and providing pulsed electromagnetic signals according to the present invention to the injection site.
Preferably apparatus and/or the transmission means or one or more electronic transmission chips are arranged in the apparatus so that the pulsed electromagnetic signals are directed to the users skin or body in use. For example, the pulsed electromagnetic signals can be directed through a first surface of the housing, and said first surface is arranged to be in direct or indirect contact with a users skin.
In one embodiment the apparatus is arranged to be implantable into a persons body. For example, the apparatus could be implanted at a site in the persons body requiring treatment. In this embodiment the apparatus is preferably an implant.
Preferably at least the outer casing of the apparatus is coated and/or formed from a material suitable for implantation into a persons body.
Preferably the attachment means includes any or any combination of a one or more straps, ties, necklaces, pendants, belts, bracelets, clips, keyrings, lanyards, VELCRO® (hook and loop fastening), press studs, buttons, button holes, adhesive, plaster, sutures, clips, bio-compatible adhesives and/or the like.
In one embodiment, the apparatus is provided with at least one holding means or reservoir for holding or containing the transfection mixture which is to be transfected into a person in use respectively.
Preferably the holding means or reservoir is arranged on the apparatus such that it is locatable on and/or adjacent to a persons skin in use. The pulsed electromagnetic signals can be directed at one or more parts of a persons body to help improve the absorption and/or transfection of the agent through the persons skin and into one or more cells of the person.
In one embodiment, it is thought that the direction of pulsed electromagnetic signals to a users skin modifies the permeability of the users skin to allow increased and/or improved take up of the transfection mixture in use. Typically, modification of the permeability of the skin occurs at least for the time period during which the pulsed electromagnetic signals are directed towards a users skin. Typically, the modification of the permeability of the users skin remains, but diminishes over time once the pulsed electromagnetic signal emission has stopped.
In one embodiment the strength and range of the pulsed electromagnetic signals is sufficient, when the housing the electronic device is located with respect to a portion of the users skin, for the pulsed electromagnetic signals to pass through the skin into the users body, and preferably at least adjacent an inner area immediately adjacent said users skin portion.
According to one aspect of the present invention there is provided a method of increasing transfection efficiency in eukaryotic cells and/or apparatus for increasing transfection efficiency in eukaryotic cells.
According to a further aspect of the present invention there is provided a method of increasing protein expression in transfected eukaryotic cells and/or apparatus for increasing protein expression in transfected eukaryotic cells.
According to one aspect of the present invention there is provided a method for providing gene therapy in vivo, said method comprising the steps of:
Preferably the method of introducing the transfection mixture into the patient includes orally, transdermally, sub-cutaneously and/or the like.
According to one aspect of the present invention there is provided a method for providing gene therapy in vitro, said method comprising the steps of:
According to a further aspect of the present invention there is provided a method of improving transfection efficiency in eukaryotic cells, said method including the steps of:
Once the patients cells have been transfected according to the method, they can then be optionally re-introduced back into the patient or another patient as required.
According to an aspect of the present invention there is provided apparatus for assisting in the provision of gene therapy in eukaryotic cells, said apparatus including a housing, transmission means located in said housing and arranged to transmit pulsed electromagnetic signals provided at any or any combination of a pre-determined frequency, at a pre-determined pulse rate, or a pre-determined power in use, control means for controlling operation of at least the transmission means in use, and power supply means for providing electrical power to the transmission means and/or control means in use.
According to a further aspect of the present invention there is provided a method of altering gene and/or protein expression, said method comprising the steps of:
In one embodiment the method kills cancer cells and increases DNA repair in healthy cells and tissue.
In one embodiment the apparatus is implantable into a patient, such as for example in a region at or adjacent cancerous tissue, to treat the cancerous tissue. This method may be useful where cancerous tissue is more distant from the patients skin.
In one embodiment the apparatus is worn by a patient at or adjacent the patients skin and could be used to deliver one or more pharmaceutical agents or drugs to cancerous tissue, such as for example located in the vicinity of a sub-dermal tumour, such as a melanoma, and/or to treat a virus.
Thus, in one embodiment, the apparatus can be used to deliver pulsed electromagnetic signals through a patients skin to interact directly with the DNA of cells to promote the apoptosis, cell of cancerous cells and/or assist in creating healthy cells to repair DNA damage.
In one embodiment the apparatus is used to deliver pulsed electromagnetic signals through a patients skin to provide an anti-viral effect.
In one aspect of the present invention there is provided a cell or progeny thereof produced using any one of the methods defined herein.
It is to be noted that reference to an improvement in transfection efficiency herein refers to an increase in the number of cells transfected and an increase or maintenance of the cell viability following a transfection process.
It will be appreciated that the present invention can be used in a laboratory based environment or can be upscaled to be used in an industrial level environment.
Specific embodiments of the invention are now described with reference to the accompanying drawings; wherein
With reference to
The apparatus 2 is in the form of an electronic device capable of emitting pulsed electromagnetic signals at a pre-determined frequency, at a pre-determined pulse rate, at a pre-determined power level and for a pre-determined period of time. The pre-determined parameters can be pre-set by the manufacturer or can be user selectable as required. The technology used in the apparatus is referred to hereinafter as the “pulsed technology according to the present invention”.
Apparatus 2 includes a housing 4. In this particular example, the housing 4 is in the form of a laboratory transfection plate, and includes a base surface 5, an upper surface 7 opposite to base surface 5, and one or more side walls 9 located between the upper and base surfaces 5,7.
Within the interior of housing 4 there is provided a circuit board 6 with an integrated circuit 8 configured and interconnected thereon to generate pulsed electromagnetic signals when operational. Control means in the form of a control unit 10 are provided to allow the selective operation of the apparatus 2. A memory device 12 is provided to allow data, one or more operating parameters, software and/or the like to be stored and retrieved when necessary. The control unit preferably includes micro-processing means to allow processing of data and/or the like.
The apparatus 2 typically also includes one or more power cells 14 to provide electrical power to the apparatus. A rechargeable facility can also optionally be provided to allow the power cells 14 to be recharged from a remote power source rather than having to be replaced.
It will be appreciated that the housing 4 may be provided in any suitable form for its intended use and can be provided with engagement means to allow the same to be located with, for example an interior or exterior of a container in which the cells to be treated are located. Alternatively, the housing may be formed as part of a container in which the cells to be treated are located. Alternatively still, the upper surface 7 can provide a planar or flat surface on which a container in which the cells are to be treated or located can be placed. Yet further still, a recess 17 could be defined in the upper surface 7 of the housing for stably supporting the placement of a container 16 in the form of, for example, a culture flask, petri dish or other culture container, so that the housing 4 is located underneath the container 16 and the container 16 is supported in the recess 17.
The integrated circuit 8 includes an electronic transmission chip that is arranged to emit the pulsed electromagnetic signals from the apparatus 2 in use. More particularly, in one embodiment of the present invention, the electronic transmission chip is arranged such that it is spaced less than 5 cm from the container 16 located in recess 17 in use, and preferably approximately 1 cm. This allows the electromagnetic signals emitted from the chip to be directed to the cells located within the container 16 in use.
The apparatus of the present invention is designed to be used at room temperature (i.e. approximately 20° C.), in temperatures colder than room temperature, such as for example in a refrigeration unit, and/or can be used at temperatures above room temperature, such as for example in an incubator unit.
In one embodiment, the control unit 10 is programmed to control the transmission chip to allow it to emit pulsed electromagnetic signals at a frequency of 2.45 GHz+/−50 MHz, at a pulsed frequency of 15 Hz and at a power of approximately 2 mW. It will be appreciated that the parameters associated with the pulsed electromagnetic signals can be adjusted and/or be user selectable as required. For example, the time for which the pulsed electromagnetic signals are emitted can be selected by the user if required. In addition, the power can be adjusted, although it typically remains in the milliwatt range so as to avoid over energising the cells contained within the container 16 in use. In one example, the pulsed signals last for 1 ms and the rest period between signals is 66 ms. This provides a duty cycle of less than 2%.
In one example, the electromagnetic signals are RF signals using the Bluetooth LE protocols advertising feature and are transmitted using GFSK between 0.45 and 0.55.
However, it should be noted that any frequency transmission in the Industrial, Medical and Scientific frequency bands (i.e. 2.4 to 2.4835 GHz, preferably 2.45 GHz+/−50 MHz) could be possible by the electronic apparatus in use.
Referring to
The six electronic chips 104 are provided a spaced distance apart in the apparatus 102. The spacing between the chips can be any required distance but, in one example, the chips are spaced apart such that when a 24 well cell plate 106 is located on upper surface 7 of the apparatus in use, one transmission chip 104 is located centrally of four of the wells. Thus, each electronic chip 102 directs pulsed electromagnetic signals to 4 wells per 24 well cell plate. An on/off operational switch 108 is provided on the apparatus 102 to move the apparatus between on and off conditions in use.
In accordance with the present invention, the apparatus as described above can be used to provide pulsed electromagnetic signals directed towards reagents and/or cells involved at one or more different stages of a transfection process. The apparatus can also be used to direct pulsed electromagnetic signals to transfected or non-transfected cells to enhance cellular protein expression. The pulsed technology of the present invention has wide and different application uses, such as gene therapy, cell transfection and/or the like as previously described.
The Applicants have undertaken experiments to show that when an agent in the form of nucleic acid, such as DNA, RNA, DNA plasmids and the like, is provided in association with an amphiphilic construct, such as a liposome vehicle, and transfected into different types of eukaryotic cells, the use of their inventive pulsed technology at different stages of the transfection process can significantly increase the transfection efficiency process and the protein expression yield.
As a simplified overview, in one example, material comprising a combined dispersion of eukaryotic cells and liposomal formulations of nucleic acid (DNA, RNA or small segments of either) is contained in a suitable container such as a culture vessel, flask or dish which, in one embodiment is located on the apparatus 2, 102 and pulsed electromagnetic signals are emitted from the apparatus and are directed through the wall of the container 16 and into the material 20.
The pulsed technology of the present invention can be used on the transfection mixture prior to transfection taking place, such as for example on the nucleic acid and/or amphiphilic constructs. The pulsed technology of the present invention can also be used, or alternatively be used, on the transfection complex including the transfection mixture and the eukaryotic cells. In addition, or alternatively still, the pulsed technology of the present invention can be used on the cells once transfection has taken place, and/or on eukaryotic cells which have not undergone transfection to increase protein expression in those cells.
In the following experiments used to exemplify the present invention, the same pulsed technology of the present invention was used on the transfection mixture prior to mixing with different eukaryotic cells lines, and/or on the eukaryotic cell lines mixed with the transfection mixture during a transfection process.
The nucleic acid used in the experiments comprised DNA plasmid material including a arginine vasopressin (AVP) promoter, a simian virus 40 (SV40) promoter, or an insulin like growth factor binding protection 3 (IGFBP3) promoter. A cytomegalovirus (Adluc) plasmid, a luciferase control vector (Renilla) plasmid or a Green Fluorescent Protein (GFP) plasmid were also used.
The amphiphilic constructs used in the experiments were either a transfection reagent containing cationic polymer (Turbofect™) (Thermo Fisher, USA), polyethylenimine (PEI) (Fisher Scientific, USA), or TransIT2020 (Mirus Bio, USA).
The cell lines used in the experiments were Chinese Hamster Ovary K1 (CHO) cells (adherent cells) (ATCC, USA-ATCC® CCL-61™), Human Embryonic Kidney (HEK) 293 freestyle cells (suspension cells) (Thermo Fisher, USA), Human Colon Tumour (HCT) 116 cells (adherent cells) (ATCC, USA-ATCC® CCL-247™) or Jurkat E6 (suspension T-cells) (ECACC), UK).
In order to determine the efficiency of the cell transfection process using the above components, the luciferase activity or the amount of green fluorescent protein was measured using suitable equipment.
The DNA plasmid material chosen was complexed with the amphiphilic construct using known techniques to form a transfection mixture. In some experiments this transfection mixture was subjected to the pulsed technology of the present invention. The transfection mixture (with or without being exposed to pulsed technology) was then mixed in a dispersion of one of the mammalian cell lines in a suitable cell culture container to form a transfection complex. This cell culture container was then placed on the apparatus housing of the present invention and subjected to the pulsed technology as previously described for a predetermined period of time. The emission of the pulsed electromagnetic signals was then stopped and the material was allowed to reach equilibrium. In addition, control experiments were also conducted using the same material and mixing requirements identically but in the absence of the pulsed technology of the present invention.
A more detailed description of the methodology used in the experiments, the results and the findings are provided below.
This experiment was undertaken to look at the effect of the pulsed technology of the present invention on the process of transfection in adherent Chinese Hamster Ovary (CHO) K1 cells (ATCC, USA) and HCT116 (Human Colon Cancer Cell Line) (ATCC, USA) using Adluc and Renilla Plasmids in either PEI (Fisher Scientific, USA) or Turbofect (Thermo Fisher, USA) amphiphilic constructs. The pulsed technology was applied to a) the cells and the transfection mixture (the transfection complex) during the transfection process only; and b) the transfection mixture prior to forming a transfection complex with the cells and then to the transfection complex during the transfection process.
Consumables
Opti-MEM™ I Reduced Serum Media (Thermo Fisher, USA)
Dulbeccos Modified Eagle Medium (DMEM) (Thermo Fisher, USA)
Fetal Calf Serum (FCS) (Hyclone, USA)
2×24 Well Plates Nunc (1.9 cm2/well) (Thermo Fisher, USA)
200 ng of AdLuc plasmid/well (Luciferase expressing plasmid/DNA) (made by Dundee University, UK)
2 ng Renilla plasmid/well (Luciferase expressing plasmid/DNA) (made by Dundee University, UK)
Alfa Aesar™ Polyethyleneimine, linear, M.W. 25.00 (PEI) (Fisher Scientific, USA)
Turbofect (Thermo Fisher, USA)
Method Steps
Control—Using PEI
1. 650 μL of Opti-MEM media was mixed with 2.6 μg of AdLuc plasmid and 26 ng of Renilla plasmid in a first tube;
2. 650 μL of Opti-MEM media was mixed with 7.88 μg of PEI in a second tube;
3. The contents of the second tube was mixed in a dropwise manner to the first tube while gently vortexing until a final volume of 1.3 mL mixture was achieved using a Vortex-Genie 2, Model G560E, (Scientific Industries, USA);
4. The transfection mixture was incubated for 15 minutes at room temperature (approx. 20° C.);
5. 100 μL of this incubated transfection mixture was then dispensed into wells labelled A1-A6 on each of the two 24 well plates (Plates 1 and 2). This formed the transfection mixture.
Invention—with Pulsed Technology Using PEI on Transfection Mixture Prior to Transfection Complex being Created
1. Then, steps 1-3 above were repeated but at step 4 the mixture forming the transfection mixture was incubated for 15 minutes at room temperature (approx. 20° C.) by locating the first tube on a pulsed electromagnetic signal device according to the present invention. The pulsed device operates as described above (i.e. pulsed device operated at 2.45 GHz+/−50 MHz, at power 2 mW using a pulsed frequency of 15 Hz).
2. 100 μL of this incubated pulsed transfection mixture was dispensed into wells labelled B1-B6 on each of the two 24 well plates (Plates 1 and 2);
Control—Using Turbofect
Luciferase Assay Protocol—Using the Dual-Luciferase Reporter Assay System (Promega, USA)
The following link sets out the protocol used but a summary of the protocol is set out below. https://www.promega.co.uk/products/luciferase-assays/reporter-assays/dual_luciferase-reporter-assay-system/?catNum=E1910#protocols
Method Steps
Table 1 shows the results of the CHO K1 cell experiments where technique 1 pulsed technology was used with the Turbofect amphiphilic construct and associated methodology.
Table 2 shows the results of the CHO K1 cells experiments where technique 2 pulsed technology was used with the Turbofect amphiphilic construct and associated methodology.
Table 3 shows the results of the HCT 116 cells experiments where pulsed technology was used with the Turbofect amphiphilic construct and associated methodology.
With reference to Tables 1 and 2 and
It can be seen that the transfection efficiency in CHO K1 cells using technique 1 pulsed technology was significantly improved compared to the control cells, with a t-test value of 0.024, an average fold increase of 1.8 and % increase of 178.0.
It can also be seen that the transfection efficiency in CHO K1 cells using technique 2 pulsed technology was significantly improved compared to the control cells, with a t-test value of 0.044, an average fold increase of 2.3 and % increase of 232.7.
Furthermore, it can be seen that experiments undertaken with technique 2 pulsed technology (i.e. the 6 electronic transmitter chip array) produced significantly better results than the experiments undertaken using technique 1 pulsed technology.
With reference to Table 3 and
It can be seen that the transfection efficiency in HCT 116 cells using pulsed technology was significantly improved compared to the control cells, with a t-test value of 0.044, a fold increase of 1.4 and % increase of 138.5.
Thus, it can be concluded that the pulsed technology of the present invention significantly increased the transfection efficiency in adherent CHO K1 cells and HCT 116 cells compared to when pulsed technology was not used. Furthermore, six electronic transmitters produced a further increase in transfection efficiency compared to where only a single electronic transmitter was used.
Experiment 2 was undertaken to look at the effect of the pulsed technology of the present invention on the process of transfection of adherent HCT116 (Human Colon Cancer Cell Line) (ATCC, USA) using the Adluc and Renilla Plasmids containing either the IGFBP3 promoter or the SV40 promoter in PEI (Fisher Scientific, USA) amphiphilic constructs. The methodology of Experiment 1 was followed for Experiment 2.
Table 4 shows the results of the HCT 116 cells experiments for the IGFBP3 promoter using the PEI amphiphilic construct and associated methodology.
Table 5 shows the results of the HCT 116 cells experiments for the SV40 promoter using the PEI amphiphilic construct and associated methodology.
With reference to Tables 4 and 5 and
It can be seen that the transfection efficiency (shown by the IGFBP3 promoter) in HCT 116 cells using pulsed technology was significantly improved compared to the control cells, with a t-test value of 0.004 and % increase of 168.5.
It can be seen that the transfection efficiency (shown by the SV40 promoter) in HCT 116 cells using pulsed technology was significantly improved compared to the control cells, with a t-test value of 0.027 and % increase of 155.2.
Thus, it can be concluded that the pulsed technology of the present invention significantly increased the transfection efficiency in adherent HCT 116 cells compared to when pulsed technology was not used.
This experiment was undertaken to look at the effect of the pulsed technology of the present invention on the process of transfection of Human Embryonic Kidney (HEK) suspension cells 293Freestyle using a green fluorescent protein (GFP) plasmid in a PEI amphiphilic construct. The pulsed technology was applied to the cells and the transfection reagent during the transfection process only.
Consumables
Opti-MEM™ I Reduced Serum Media (Thermo Fisher, USA)
Green Fluorescent Protein (GFP) plasmid (made by Dundee University, UK)
293-Freestyle Suspension Cells (Thermo Fisher, USA)
293-Free Expression Media (Sigma-Aldrich, USA)
Alfa Aesar™ Polyethyleneimine, linear, M.W. 25.00 (PEI) (Fisher Scientific, USA)
Method Steps when Pulsed Technology Used on Reagent and Cell Mixture Only
With reference to
It can be seen that the transfection efficiency (shown by the amount of the mean Green Fluorescence measured) in HEK 293 Freestyle Suspension Cells using pulsed technology was significantly improved compared to the control cells, with a t-test value of less than 0.05 and a peak increase of 2.3 fold more GFP expression was observed, as shown in
It can be seen that the transfection efficiency (shown by amount of the mean Green Fluorescence measured) in HEK 293 Freestyle Suspension Cells using pulsed technology was significantly improved compared to the control cells, with a t-test value of less than 0.05 and an over 50% increase in GFP expression was observed, as shown in
Thus, it can be concluded that the pulsed technology of the present invention significantly increased the transfection efficiency in HEK 293 Freestyle Suspension Cells compared to when pulsed technology was not used.
This experiment was undertaken to look at the effect of the pulsed technology of the present invention on the process of transfection in Jurkat E6 Cells (Human leukaemic T-Cell lymphoblast cells) (European Collection of Authenticated Cell Cultures (ECACC), UK) using Adluc and Renilla Plasmids in either PEI (Fisher Scientific, USA) or TransIT2020 (Mirus Bio, USA) amphiphilic constructs. The pulsed technology was applied to a) the cells and the transfection mixture (the transfection complex) during the transfection process only; and b) the transfection mixture prior to forming a transfection complex with the cells and then to the transfection complex during the transfection process.
Consumables
Opti-MEM™ I Reduced Serum Media (Thermo Fisher, USA)
Fetal Calf Serum (FCS) (Hyclone, USA)
RPMI Medium (Sigma-Aldrich, UK)
2×24 Well Plates Nunc (1.9 cm2/well) (Thermo Fisher, USA)
1 μg of AdLuc plasmid/well (Luciferase expressing plasmid/DNA) (made by Dundee University, UK)
80 ng Renilla plasmid/well (Luciferase expressing plasmid/DNA) (made by Dundee University, UK)
Alfa Aesar™ Polyethyleneimine, linear, M.W. 25.00 (PEI) (Fisher Scientific, USA)
TransIT2020 (Mirus Bio, USA)
Method Steps
Control—Using PEI
1. 650 μL of Opti-MEM media was mixed with 13 μg of AdLuc plasmid and 1 μg of Renilla plasmid in a first tube;
2. 650 μL of Opti-MEM media was mixed with 42 μg of PEI in a second tube;
3. The contents of the second tube was mixed in a dropwise manner to the first tube while gently vortexing until a final volume of 1.3 mL mixture was achieved using a Vortex-Genie 2, Model G560E, (Scientific Industries, USA);
4. The transfection mixture was incubated for 15 minutes at room temperature (approx. 20° C.);
5. 100 μL of this incubated transfection mixture was then dispensed into wells labelled A1-A6 on each of the two 24 well plates (Plates 1 and 2). This formed the transfection mixture.
Invention—with Pulsed Technology Using PEI on Transfection Mixture Prior to Transfection Complex being Created
1. Then, steps 1-3 above were repeated but at step 4—the mixture forming the transfection mixture was incubated for 15 minutes at room temperature (approximately 20° C.) by locating the first tube on a pulsed electromagnetic signal device according to the present invention. The pulsed device operates as described above (i.e. pulsed device operated at 2.45 GHz+/−50 MHz, at power 2 mW using a pulsed frequency of 15 Hz).
2. 100 μL of this incubated pulsed transfection mixture was dispensed into wells labelled B1-B6 on each of the two 24 well plates (Plates 1 and 2);
Control Using TransIT2020
Cell Lines Added to Plates 1 and 2
Luciferase Assay Protocol—Using the Dual-Luciferase Reporter Assay System (Promega, USA)
Method Steps—as Set Out Above
Table 6 shows the results of the Jurkat E6 cells experiments for the AdLuc and Renilla Plasmids using the PEI or TransIT2020 amphiphilic constructs and associated methodology.
Exp A— where pulsed technology was applied to the transfection complex only (i.e. once the transfection mixture had been added to the cells and during incubation).
Exp B—where pulsed technology was applied to the transfection mixture (prior to adding the Jurkat E6 Cells) only.
Exp C—where pulsed technology was applied to the transfection mixture prior to adding the Jurkat E6 Cells) and then also to the transfection complex (i.e. once the transfection mixture had been added to the cells and during incubation).
With reference to Table 6 and
In one embodiment of the present invention, as shown in
The apparatus 301 is capable of emitting pulsed electromagnetic signals at a pre-determined frequency, at a pre-determined pulse rate, at a pre-determined power level and for a pre-determined period of time as previously described. However, this apparatus 301 can be worn adjacent a patients body to allow the pulsed electromagnetic signals to be directed towards the patients body in use. The pre-determined parameters can be pre-set by the manufacturer or can be user selectable as required.
The apparatus 301 includes a housing 302, which includes a pulsed signal transmission system. In particular, in this example, the pulsed signal transmission system includes a circuit board 307 with transmission means in the form of an electronic transmission chip 304, typically provided as part of an integrated circuit, which allows the transmission of pulsed electromagnetic signals when the device is operational in use.
In one example, the housing includes a base surface 303, an upper surface 311 opposite to base surface, and one or more side walls 313 located between the upper and base surfaces 311, 303 respectively.
Control means in the form of a control unit 310 can be provided to allow the selective operation of the apparatus 301. A memory device 306 is provided to allow data, one or more operating parameters, software and/or the like to be stored and retrieved when necessary. The control unit preferably includes micro-processing means to allow processing of data and/or the like.
The apparatus 301 could also include one or more power cells 310 to provide electrical power to the apparatus. A rechargeable facility can also optionally be provided to allow the power cells to be recharged from a remote power source rather than having to be replaced.
The electronic transmission chip 304 is arranged in the housing 302 to emit the pulsed electromagnetic signals from the apparatus 301 in a particular direction or directions use. The direction of transmission of the pulsed electromagnetic signals will typically depend on what purpose the apparatus 1 is being used for. If the apparatus is being used for wearing by a user, the signals are typically directed through base surface 303 towards the user.
In one embodiment of the present invention, the electronic transmission chip is arranged in the housing 302 such that it is spaced less than 5 cm from the surface of the housing 302 that is to be brought into contact with a users skin in use, and preferably approximately 1 cm. This allows the electromagnetic signals emitted from the chip to be directed to the patient in use.
The apparatus of the present invention is designed to be used at room temperature (i.e. approximately 20° C.), in temperatures colder than room temperature and/or can be used at temperatures above room temperature, such as for example in a patients body.
In one embodiment, the control unit 310 is programmed to control the transmission chip to allow it to emit pulsed electromagnetic signals at a frequency of 2.45 GHz+/−50 MHz, at a pulsed frequency of 15 Hz and at a power of approximately 2 mW. It will be appreciated that the parameters associated with the pulsed electromagnetic signals can be adjusted and/or be user selectable as required. For example, the time for which the pulsed electromagnetic signals are emitted can be selected by the user if required. In addition, the power can be adjusted, although it typically remains in the milliwatt range so as to avoid over energising the cells contained within the container 16 in use. In one example, the pulsed signals last for 1 ms and the rest period between signals is 66 ms. This provides a duty cycle of less than 2%.
However, it should be noted that any frequency transmission in the Industrial, Medical and Scientific frequency bands (i.e. 2.4 to 2.4835 GHz, preferably 2.45 GHz+/−50 MHz) could be possible by the electronic apparatus in use.
In one example, the electromagnetic signals are RF signals using the Bluetooth LE protocols advertising feature and are transmitted using GFSK between 0.45 and 0.55. However, it should be noted that any frequency transmission in the Industrial, Medical and Scientific frequency bands could be possible by the electronic apparatus in use.
In the illustrated example in
In the embodiment shown in
In another embodiment of the present invention, as shown in
In another embodiment of the present invention, as shown in
In yet further embodiment of the present invention, as shown in
In one example, the apparatus of the present invention could be worn so as to minimise viral replication and as a means to provide greater immunological protection to the wearer. Thus, in this embodiment, when the pendant 336 is worn at the level of throat/upper chest, a boost is provided to the immunity of this critical respiratory zone in the wearer.
Typically, in whichever embodiment, the apparatus of the present invention is provided at or adjacent a portion of the skin of a user which has been selected to provide a topical and focused treatment at a predetermined location.
For example, if the purpose of the apparatus is to provide a treatment for a cancerous tumour in a patient, the apparatus is located in the vicinity of, or is implanted into, a recognised cancerous tumour such as may be present, for example, in the liver, kidney, breast or bone. Alternatively, if the apparatus is to be provided to achieve a therapeutic benefit or to limit or prevent the possibility of infection, the apparatus can be located externally of the patient adjacent the portion of the patients body at which therapeutic or preventative effect is believed to be most beneficial, such as at the throat region of the patient or person.
Thus, if the apparatus is located directly on the skin 312 of a patient, the pulsed electromagnetic signals are emitted through the skin and into the tumour to provide a change in condition of the tumour cells. If the apparatus is to be used in conjunction with a patch or other drug carrying item, such as for example as shown in
Although the above examples shows transfection of an agent in the form of nucleic acid associated with an amphiphilic construct in different types of eukaryotic cells being significantly improved following exposure to the pulsed technology of the present invention at different stages of the transfection process, the Applicants fully expect and predict that the transfection and/or intra-cellular delivery of one or more pharmaceutical and/or therapeutic agents or compounds, small molecules or small molecular material of less than 5 Kilodaltons, large molecules or large molecular material of greater than or equal to approximately 5 Kilodaltons, one or more proteins, vaccine, an organic agent, and/or one or more antibodies when associated with an amphiphilic construct, to be significantly improved on exposure of the same to the pulsed technology of the present invention in one or more eukaryotic cells. These predictions and expectations are based on data already collected by the Applicants in their co-pending application claiming priority from British Patent Applications GB2004411.1, GB2009297.9, GB20044112.9 and GB2009296.1, the content of which is incorporated herein by reference, which shows that the intra-cellular delivery of a “naked agent” in the form of Doxorubicin (not associated with an amphiphilic construct) in eukaryotic cells is significantly improved when exposed to the pulsed technology of the present invention. The data for these experiments is reproduced below to show support for the breadth of the claim set of the present application. The Applicants predict the same or similar mechanism of improvement of transfection efficiency and/or intra-cellular delivery when an agent is associated with an amphiphilic construct as when a “naked agent” (i.e. not associated with an amphiphilic construct) is used. This is because the pulsed electromagnetic waves or signals according to the present invention are thought to be sufficient to rotate H2O periodically around its dipole with relatively long rest or relaxation periods. The periodic rotation of H2O is thought to interrupt hydrogen bonding in the phospholipid bilayer or cell membranes of the eukaryotic cells. This periodic or intermittent low energy perturbation of the cell membranes is thus thought to stimulate increased interaction with the agent, some molecules and/or cell membranes and their environment, such as for example, the nucleic acid or agent with the cell membrane. The relatively long rest or relaxation period between the pulses of the pulsed electromagnetic signals is thought to be sufficient to maintain cellular integrity.
In the following experiments taken from the Applicants co-pending patent application, the same pulsed technology of the present invention was used on a “naked agent” in the form of Doxorubicin when added to a eukaryotic cells line.
Human Colon Tumour (HCT) 116 cells (adherent cells) (ATCC, USA-ATCC® CCL-247™) were seeded at a density of 3×105 cells per well in two CELLSTAR®6-well plates (9.6 cm2) in a final volume of 5 mL Dulbeccos Modified Eagle Medium (DMEM) (Thermo Fisher, USA)+10% Fetal Bovine Serum (FBS) (Hyclone, USA) 24 hours before treatment.
The naked agent used was Doxorubicin (0.25 μM) (Sigma Aldrich) in absolute ethanol and was given to the cells for a 1 hour treatment period and incubated at 37° C., at 5% CO2.
After treatment the media was removed and fresh media was added to the cells. One of the plates was incubated directly at 37° C., at 5% CO2 and the second plate was placed in a different incubator and pulsed using the pulsed technology of the present invention at 37° C., at 5% CO2.
Protein extracts were collected at 3 hours, 6 hours, 9 hours, 16 hours or 24 hours of treatment for analysis by SDS-page.
The following Western Blot protocol is set out in reference [5].
Preparation of Protein Extracts for Western Blot
1. For protein extraction the cells were washed twice with ice-cold PBS and then lysed in NP-40 extraction buffer (50 mM Tris ph 7.5; 10% glycerol; 0.1% “NP-40 Alternative” (Merck Millipore, USA); 100 mM NaCl; 0.2 mM EDTA) supplemented with 1× Complete™ Protease Inhibitor Cocktail (Roche, Switzerland). Extracts were sonicated (20 seconds, 20% amplitude) and protein concentration was determined using BCA™ Protein Assay Kit (ThermoFisher Scientific USA) according to the manufacturers recommendations.
Western Blot Protocol
1. Protein extracts (15/20 μg depending on the experiment) were supplemented with 0.1M dithiothreitol (DTT) and 1×LDS buffer (Invitrogen, USA) and were heated at 95° C. for 10 min before loading on NuPAGE 10% Bis-Tris polyacrylamide gels (Invitrogen, USA).
2. Protein samples were separated by electrophoresis (100V) using 1×MOPS Running Buffer. Transfer of proteins was performed at 12V overnight onto a nitrocellulose membrane (Protran 0.1 μm from GE Healthcare, USA) in 1× Transfer Buffer supplemented with 20% methanol. 1× Transfer Buffer is prepared from 10× Wet blot solution containing 144 g of glycine and 30 g Tris-Base in a final volume of 1 L milli-Q water.
3. Membranes were blocked for 30 min in 5% BSA diluted in PBS—0.1% Tween20 before being incubated overnight with a primary antibody (Mouse monoclonal antibody D01). After a wash of 15 min. in PBS-Tween20, membranes were incubated for 1 h with a corresponding secondary antibody (HRP conjugated Donkey anti Mouse). All secondary antibodies, conjugated with Horse Radish Peroxidase (HRP), were purchased from Jackson ImmunoResearch lab and used at 1:10000/1:15000 dilution (depending on the antibody) in 5% BSA PBS-Tween20.
At the end of the incubation membranes were washed twice with PBS-Tween20 for 15 min followed by a final 10 min. wash with PBS. The chemiluminescence signal was detected on Hyperfilm™ ECL (Cytiva, USA) using the Amersham ECL Western Blotting Detection System (Cytiva, USA).
Results
Referring to H2AX was used as a marker to ensure that if any effect was observed it was not caused due to ionising radiation.
H2AX's expression changes when ionising radiation is present, and since there is no observed change between the pulsed technology according to the present invention and the control arms, it was concluded that the pulsed technology of the present invention did not emit ionising radiation.
Ku80 was used as the loading control to ensure that equal concentrations of each sample was loaded onto each well. Equal concentrations of Ku80 make the rest of the bands in the Western Blot comparable.
Referring to
In conclusion, there is clear evidence that treating the cells with the pulsed technology according to the present invention increases the ability of the cells to uptake doxorubicin from the media as various p53 isoforms were upregulated more in the pulsed technology arm compared to the control arm. It can be concluded that this effect is not caused by ionising radiation as the radiation marker gH2AX remained unchanged between the pulsed technology arm and the control arm.
Therefore, the combined effect of enhanced delivery of anti-cancer drugs and the direct treatment of pulsed technology according to the present invention affects beneficially the regulation of replication via the p53 oncogene and improves cancer treatment. Moreover, the effect of the pulsed technology of the present invention on non-mutated p53 of healthy cells results in increased repair of these cells.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2004411.1 | Mar 2020 | GB | national |
| 2004412.9 | Mar 2020 | GB | national |
| 2009296.1 | Jun 2020 | GB | national |
| 2009297.9 | Jun 2020 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/GB2021/050737 | 3/25/2021 | WO |
